The respiratory tract is a major portal for pathogens. The bronchoalveolar macrophage, positioned at the mucosal surface, recognizes "pathogen associated molecular patterns (PAMPs)" through structures called "pattern recognition receptors (PRRs)." A family of closely related mammalian PRRs, the "Toll-like receptors (TLRs)," are transmembrane signaling molecules that respond to diverse PAMPs. The lipopolysaccharide (LPS) of Gram negative bacteria stimulates macrophages through TLR4 to elicit a pattern of gene expression that is strongly proinflammatory, e.g., TNF-a, IL-1[unreadable], IL-12, and IFN-? resulting in a "Th1-type" cytokine milieu. Respiratory syncytial virus (RSV) is the leading cause of pneumonia and bronchiolitis in infants and young children worldwide, and has recently been attributed to increased morbidity and mortality in the elderly and immunosuppressed. In a failed clinical trial, a formalin-inactivated RSV (FI-RSV) vaccine led to exacerbated RSV disease. Vaccine-enhanced RSV disease, demonstrated by histopathology and airway hyperreactivity, are faithfully recapitulated in the cotton rat (S. hispidus), and is associated with a Th2-type pattern of cytokine gene expression. Vaccination with FI-RSV and a TLR4 agonist mitigates enhanced disease. Recently, the RSV fusion (F) protein was shown to be a TLR4 agonist. In vitro, cotton rat macrophages respond to LPS and F protein to elicit the same spectrum of inflammatory genes, although the F protein is less potent. The overall hypothesis to be tested is that the interaction of F protein with TLR4 is essential for establishment of a Thl-type milieu and that formalin fixation of RSV destroys the capacity of F to interact with TLR4, resulting in a Th2-type response. This proposal details novel experimental approaches: (i) to evaluate the contribution and regulation of F protein signaling through TLR4 to the development of a Th1-type cytokine profile, (ii) to determine the role of TLR4 in the development of RSV-mediated disease in vivo, and (iii) to determine if polymorphic forms of TLR4 previously associated with airway hyporesponsiveness to LPS exhibit diminished sensitivity to F protein and if they are overrepresented in a population at high risk for RSV. It is expected that at the completion of this grant, we will have determined the molecular basis for primary and vaccine-enhanced RSV disease, and have identified strategies for the development of a human RSV vaccine. [unreadable] [unreadable]